Atherosclerosis is believed to be an inflammatory disease, and accumulating evidence supports a possible role of microbial products, and the receptors that they activate, in the inflammatory process. Whereas most research has focused on the promflammatory roles of macrophages, research by our laboratory and others has shown that vascular smooth muscle cells (VSMC) can be infected by intracellular bacteria and activated by microbial products, whereupon they produce proinflammatory cytokines and chemokines, and proliferate. The long-term objective of this project is to determine the roles of interleukin 1 (IL-1) and Toll-like receptors (TLR) in promoting arterial inflammation and neointima formation in vivo. TLRs are transmembrane receptors known to be essential in microbial detection and activation of innate immunity. During the preceding grant we found that human and mouse VSMC express TLR2, TLRS, and TLR4, and that activation of these receptors by low levels of their respective microbial agonists (Chlamydia pneumoniae, double-stranded RNA (dsRNA), and E. coli lipopolysaccharide (LPS)) induces early and robust expression of monocyte chemoattractant protein 1 (MCP-1). Also, LPS and dsRNA increase intranuclear IL-la levelsand stimulate proliferation in human coronary artery SMC. Preliminary findings suggest that endogenous IL-la interacts with necdin, an intranuclear growth suppressor, in human VSMC, and that IL-la and necdin may reciprocally regulate human VSMC proliferation. Therefore, the proposed studies will use selective TLRS and TLR4 agonists in concert with molecular and genetic approaches to determine the mechanisms involved in TLR-induced cellular proliferation in vitro and in vivo. Three hypotheses will be tested: 1) that TLRS- and TLR4-induced VSMC proliferation involves IL-la, and its receptor-independent inhibition of the suppressive effects of necdin on cell growth; 2) that dsRNA stimulates proliferation of VSMC by a cell type-specific pathway involving not only TLRS and the adapter molecule TRIP, but also a novel dependence on MyD88; and 3) that TLR4 expressed by resident arterial cells contributes to neointima formation and chemokine expression in vivo. The specific aims are: to determine whether endogenous IL-la promotes VSMC proliferation, and whether this action is mediated by its interactions with necdin and/or with the type I IL-1 receptor; to determine whether TLRS and MyD88-dependent ERK1/2 activation are involved in mediating dsRNA-induced proliferation in VSMC; and to determine whether TLR4 contributes to injury- induced chemokine expression and neointima formation using an endothelial denudation injury model in hypercholesterolemic Apolipoprotein E-deficient mice, and to determine the role of TLR4 expressed by bone marrow-derived and resident arterial cell populations to these responses, by using TLR4 chimeric mice. These studies will provide new insights into the mechanisms whereby microbial agents and TLR ligands in the arterial wall may promote proatherogenic arterial inflammation and cellular proliferation, and thereby may provide new molecular targets for pharmacotherapeutic intervention in human vascular disease.